The process of growth and proliferation of eukaryotic cell includes that the parent cell produces two identical daughter cells through the mitosis of the cell chromosome by accurately replicating its genome containing genetic information. This process of cell proliferation and division is called the cell cycle, and it involves the process of a cell going from one division to the next. The cell cycle consists of four growth stages: the G1 phase of massive synthesis of proteins and RNA after mitosis, the S phase of DNA synthesis and replication, the G2 phase of preparation before mitosis, and the M phase of mitosis. Cells divide and proliferate through the cell cycle, or stop, depending on the state and needs of the cell. It is necessary to keep genetic information complete and correct during cell proliferation and division. Whether or not to enter the next phase of cell cycle until the completion of the whole cell cycle is ensured and completed through the checkpoints in the cell cycle process.
During the whole process of cell cycle, there are many cell cycle checkpoints. Each cell cycle checkpoint consists of a very complex system and is composed of multiple factors. In the G1 phase, the checkpoint determines whether to enter the cell cycle by examining the state inside and outside the cell, so as to determine whether the cell enters the S phase of DNA synthesis. The G1 checkpoint is a complex system that includes the famous CDK4/CDK6. Another important checkpoint is the so-called G2-M checkpoint, where the cell completes DNA replication (S phase) and enters the cell growth phase (G2 phase). This checkpoint examines whether there is any DNA damage or defect after the cells have synthesized DNA, which determines whether the cells undergo mitosis (M-phase) with the separation of the following chromosomes. Cell cycle checkpoints at this stage include complex kinase Cdk1 complexes including Cyclin-B-cdc2 (Nurse, P., 1990, Nature 344, 503-508). Activation of Cdk1 leads to initiation of mitosis, and subsequent inactivation is accompanied by the completion of mitosis. The activity of Cdk1 is regulated by cdc2 binding to Cyclin-A or Cyclin-B and its phosphorylation. For example, the activation of the cyclin B-Cdk1 complex causes mitosis (Lindqvist, A., et al, 2009, The Journal of cell biology 185, 193-202). Cdc2 is kept inactive by phosphorylation before mitosis. Its phosphorylation state is achieved by tyrosine kinase Wee1, etc. In addition, there are M-phase cell cycle checkpoints.
Tyrosine 15 (Y15) on Cdk1 is phosphorylated by Wee1, thus inhibiting the activity of Cdk1 (McGowan, C. H., et al, 1993, The EMBO journal 12, 75-85; Parker, L. L., et al, 1992, Science 257, 1955-1957). Therefore, Wee1 is a key inhibitory regulator of Cdk1 activity and plays an important role in G2-M phase checkpoints to ensure the entry into mitosis without DNA damage after DNA replication (O'Connell, et al, 1997, The EMBO journal 16, 545-554). Loss or inactivation of Wee1 may result in premature entry into mitosis, leading to mitotic failure and cell death (Stumpff, J., et al, 2004, Curr Biol 14, 2143-2148). Some tumor cells have functional deficiency in G1 cell cycle checkpoint and rely on G2-M cell cycle checkpoints to ensure the progress of cell cycle (Sancar, A., et al, 2004, Annual review of biochemistry 73, 39-85). Due to the loss of p53 protein function, in these cancer cells, the loss of Wee1 expression or the inhibition of Wee1 activity will result in the loss of G2-M phase checkpoints, making tumor cells very sensitive to DNA damage, and this sensitivity is especially prominent in tumor cells that lose the ability of G1 phase checkpoint (Wang, Y., et al, 2004, Cancer biology & therapy 3, 305-313).
In summary, inhibition of Wee1 activity can selectively promote the death of cancer cells with defective cell cycle checkpoints; at the same time, has little effect on normal cells with normal cell cycle checkpoints. Therefore, Wee1 inhibitors may be used as targeted drugs for the treatment of cancer and other cell proliferation disorders.
In addition, because the inhibition of Wee1 activity increases the sensitivity of cells to DNA damage, Wee1 inhibitors can be used in combination with anticancer drugs that cause DNA damage or inhibit DNA repair mechanism, including PARP inhibitors, e.g. Olaparib, Niraparib, Rucaparib and Talazoparib; HDAC inhibitors, e.g. vorinotat, lomidacin, pabista, and belistatin; and the like, for treating cancer or other cell proliferation disorders. Wee1 inhibitors may also be used in combination with other anticancer drugs related to cell cycle checkpoints of cell division, including Chk1/2 inhibitors, CDK4/6 inhibitors such as Paboxini, ATM/ATR inhibitors etc. for the treatment of cancer and other diseases.
The study of Karnak et al. (Clin Cancer Res, 2014, 20(9): 5085-5096) shows that the combination of Wee1 inhibitor AZD1775 and PARP inhibitor olaparib can enhance the sensitivity of pancreatic cancer after radiotherapy. The results confirmed that the combination of Wee1 inhibitor and PARP inhibitor could enhance the radiosensitivity of pancreatic cancer, and supported the hypothesis that Wee1 inhibition could sensitize the cell to PARP inhibitor, i.e., sensitize the cell to radiotherapy by inhibiting the function of DNA repair and G2 checkpoint. It can eventually lead to the accumulation of unrepaired damaged DNA until the cell dies.
In addition, it was reported (BMC Cancer, 2015, 15: 462) that Wee1 inhibitor MK1775 and Chk1/2 inhibitor AZD7762 were used together in malignant melanoma cell and xenograft models. The results showed that the combined use of Wee1 and Chk1/2 inhibitors could synergize the inhibitory effect of single drug, thus reducing the proliferation capacity of tumor cells and activating the apoptosis mechanism. The combination of both inhibitors can inhibit tumor growth better in the xenograft model.
AZD1775 is the first Wee1 kinase inhibitor with single antitumor activity in a preclinical model. Phase I clinical studies showed the single drug efficacy of AZD1775 in patients with solid tumors with BRCA mutations, and the inhibition mechanism of Wee1 kinase was confirmed by paired tumor biopsy finding changes related to targeting and DNA damage response (J Clin Oncol, 2015, 33: 3409-3415). In a clinical phase I trial of AZD1775, which enrolled in more than 200 patients, the efficacy of AZD1775 alone or in combination with gemcitabine, cisplatin or carboplatin in the treatment of patients with advanced solid tumors was studied, showing that AZD1775 alone or in combination with chemotherapy was safe and tolerable at a certain dose. Of 176 evaluable patients, 94 (53%) had stable disease as the best response, and 17 (10%) had partial response. Importantly, the response rate of AZD1775 in patients with TP53 mutation (n=19) was 21%, while that in TP53 wild-type patients (n=33) was 12%, showing great potential for patients with TP53 mutation (J Clin Oncol, 2016 Sep. 6, pii: JCO675991).
WO2012161812 disclosed the following tricyclic compounds as Wee1 kinase inhibitors, wherein, X is N or CR1; Y is N or CR2; Z is O, S or NH; R1 and R2 are H or C1-6 alkyl; R3 is C1-8 alkyl, C2-8 alkenyl, C3-8 cycloalkyl, aryl, or heteroaryl etc; R4 is phenyl, naphthyl, tetrahydronaphthyl, indenyl or indanyl, or 5-16 member monocyclic, bicyclic or tricyclic heterocyclic groups, etc.

WO2005021551 disclosed the following tetracyclic pyrimidine or pyridine compounds as protein kinase inhibitors, wherein, X is N or CH; Y is NH, N(CN), O or S; L is a 4-atom chain made up of C and N atoms; Ra is H, C1-8 alkyl, CN, phenyl or benzyl; R1 and R2 are independently substituted saturated or unsaturated 5-, 6-, or 7-member monocyclic group, or 6-, 7-, 8-, 9-, 10- or 11-member bicyclic group (including 0, 1, 2, 3 or 4 atoms selected from N, O and S, of which O and S atoms do not exist at the same time, and the C atoms in the ring are substituted by 0, 1 or 2 oxygen groups) etc.
